JP6400473B2 - Addition of iron to improve cell culture - Google Patents

Description

RELATED APPLICATION This application claims priority to US Provisional Application No. 61 / 550,058, filed Oct. 21, 2011, the contents of which are hereby incorporated by reference in their entirety. is there.

  A common problem in mammalian cell culture is cell death at the end of the culture, which can be caused by various injuries such as nutrient depletion, inhibitor accumulation, and / or oxidative stress, among others. . Maintain cell viability and / or delay cell death over the duration of the cell culture process, generally and especially for those who utilize mammalian culture systems to express pharmaceutically relevant protein products That is a big concern. Several methods have been used to increase the viability of cell cultures, including the use of media additives and overexpression of anti-apoptotic genes. See, for example, Arden et al., “Chemical caspase inhibitors enhance cell culture viabilities and protein titer”, Biotechnol Prog. , March-April, 2007, 23 (2): 506-511, Mastrangelo et al., "Anantioptochemical chemicals proponents of biological cells upside biosindrivon sindbion sindbion 1999, November 5, 65 (3): 298-305, Balcarcel et al., "Rapamycin Reduces Hybridoma Cell Death and Enhancing Monoclonal Antibiotic Production, 1, Biotechnol Biol. “The growth factor inhibitor suramine reduce apoptosis and cell aggregation in protein-free CHO cell batch cultures.” Biotechnol. Prog. , 2000, 16, 319-325, Simpson et al., “Prevention of hybridoma cell death by bcl-2 duling subcultural conditions,” Biotechnol Bioeng, 1997, 54: 1-16, v, 1-16, sev, et al. overexpression of the apoptosis suppressor gene bcl-2. ", Biotechnol Bioeng. , 1996, 52: 166-175, Mastrangelo et al., “Overexpression of bcl-2 family members enhances in 2000, and enumeration of biologics in biologics. , "Inhibiting the apoptosis pathway using MDM2 in mammalian cell cultures.", Biotechnology and Bioengineering, 2007, Vol. 97, 601-614). However, in some cases, the use of media additives may delay cell cycle progression, resulting in lower cell density and thus lower productivity. Some media additives can protect cells from apoptosis during the growth phase but are not effective during the death phase. In addition, most media additives are expensive and may not be practical for large scale pharmaceutical manufacturing. With overexpression of anti-apoptotic genes, gene transfection is a challenge and the effects can be situation dependent.

  Thus, there is a need to improve cell culture to increase cell viability, density and / or titer.

  The present invention unexpectedly adds iron, particularly high concentrations of iron, to the cell culture medium that can significantly improve the cell density, viability and / or titer of the culture, among other advantages. Includes the discovery of Thus, the present invention provides an effective but inexpensive and easy solution for improving cell culture. The present invention is particularly useful for improving viability at the end of long-term cell culture.

  In one aspect, the invention provides a cell (eg, a product of a cell culture process) in a cell culture medium for initiating the cell culture process, and a concentration of iron in the cell culture medium over the period of the cell culture process. Adding a composition comprising iron to the cell culture medium during the cell culture process such that the cell density, viability, and / or titer in the cell culture is increased. To do.

According to the present invention, the iron-containing composition can be selected from various iron-containing compounds. In some embodiments, compositions containing iron, FeSO _4, citric acid Fe, Fe- transferrin, chloride Fe, nitrate _{Fe, Fe-EDTA, Fe (} NO 3) 3, FeCl 2, FeCl 3 and its Selected from the group consisting of combinations.

  In some embodiments, inventive methods according to the invention include adding a composition comprising iron at various points throughout the cell culture process. In some embodiments, the composition comprising iron is added after day 0. In some embodiments, the composition comprising iron is added on day 1 or later. In some embodiments, the composition comprising iron is added on or after day 3. In some embodiments, the composition comprising iron is added on day 6 or later. In some embodiments, the composition comprising iron is added on or after day 9. In some embodiments, the composition comprising iron is added at multiple points during the cell culture process.

  In some embodiments, the concentration of iron in the cell culture medium (eg, after one or more additions of a composition comprising iron) ranges between 100 μM and 5 mM. In some embodiments, the concentration of iron in the cell culture medium ranges between 300 μM to 1 mM. In some embodiments, the concentration of iron in the cell culture medium is 1 mM.

  A variety of cell types may be used in accordance with the present invention. For example, in some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a BALB / c mouse myeloma line, human retinoblast (PER.C6), monkey kidney cell, human embryonic kidney line (293), baby hamster kidney cell (BHK). Chinese hamster ovary cells (CHO), mouse Sertoli cells, African green monkey kidney cells (VERO-76), human cervical cancer cells (HeLa), canine kidney cells, buffalo rat hepatocytes, human lung cells, human hepatocytes , Mouse breast cancer cells, TRI cells, MRC5 cells, FS4 cells, or human hepatocellular carcinoma line (Hep G2). In some embodiments, the mammalian cell is a CHO cell.

  In some embodiments, the cell culture process is a fed-batch process. In some embodiments, the cell culture process is a batch-refeded culture process. In some embodiments, the cell culture process is a perfusion culture process. In some embodiments, the cell culture process is a large scale production culture process. In some embodiments, the volume of the cell culture medium is at least about 500L. In some embodiments, cell culture is performed in shake flasks.

  In some embodiments, auxiliary proteins are added to improve cell culture density, viability and / or titer. In some embodiments, the cell culture medium does not contain a hydrolyzate. In some embodiments, the cell culture medium contains a hydrolyzate.

  In some embodiments, the cell carries a gene encoding a recombinant protein. In some embodiments, the recombinant protein is an antibody or fragment thereof. In some embodiments, the antibody is an anti-IL-22 antibody. In some embodiments, the antibody is an anti-GDF8 antibody. In some embodiments, the antibody is a Myo29 antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the single domain antibody is an anti-TNF Nanobody. In some embodiments, the recombinant protein is a glycoprotein. In some embodiments, the recombinant protein is a therapeutic protein. In some embodiments, the therapeutic protein is an antibody, growth factor, clotting factor, cytokine, fusion protein, drug substance, vaccine, enzyme, or Small Modular ImmunoPharmaceutical ™ (SMIP). In some embodiments, the methods of the invention described herein further comprise purifying the recombinant protein.

  In another aspect, the present invention provides a recombinant protein produced from cells cultured according to the methods of the invention described herein.

  In yet another aspect, the invention provides a method of preventing or delaying cell death in a cell culture by adding a composition comprising iron at one or more times following the beginning of the cell culture process.

  In another aspect, the invention provides a method of inhibiting apoptosis in a cell culture by adding a composition comprising iron at one or more times following the beginning of the cell culture process.

  In some embodiments, the composition comprising iron is added after day 0. In some embodiments, the composition comprising iron is added on or after day 3. In some embodiments, the composition comprising iron is added on day 6 or later. In some embodiments, the composition comprising iron is added on or after day 9.

  In some embodiments, iron is added in an amount such that the concentration in the cell culture medium ranges between 100 μM to 5 mM. In some embodiments, iron is added in an amount such that the concentration in the cell culture medium ranges between 300 μM and 1 mM. In some embodiments, iron is added in an amount such that the concentration in the cell culture medium is 1 mM.

  In some embodiments, the cell culture is a fed-batch culture. In some embodiments, the cell culture is a rebatch culture. In some embodiments, the cell culture is a perfusion culture. In some embodiments, the cell culture is a large scale production culture. In some embodiments, the volume of the cell culture is at least about 500L. In some embodiments, cell culture is performed in shake flasks.

In some embodiments, the composition comprising iron, FeSO _4, citric acid Fe, Fe- transferrin, chloride Fe, nitrate _{Fe, Fe-EDTA, Fe (} NO 3) 3, FeCl 2, FeCl 3 , and combinations thereof Selected from the group consisting of

  In yet another aspect, the present invention provides a kit for making a cell culture medium comprising, inter alia, one or more reagents for making an initial cell culture medium and a separate composition comprising iron. provide. In some embodiments, the kit comprises a separate composition comprising iron at one or more times during the cell culture process, eg, to increase cell density, viability, and / or titer. Includes instructions to add to the initial cell culture medium.

  In some embodiments, the separate composition comprising iron is in an amount such that the iron concentration ranges between 100 μM and 5 mM in the initial cell culture medium. In some embodiments, the separate composition is such that the iron concentration ranges between 100 μM and 5 mM in at least a 500 L cell culture.

In some embodiments, a separate composition, FeSO _4, citric acid Fe, Fe- transferrin, chloride Fe, nitrate _{Fe, Fe-EDTA, Fe (} NO 3) 3, from FeCl 2, FeCl _3, and combinations thereof Selected from the group consisting of

  In some embodiments, the kit further comprises auxiliary components for fed-batch culture. In some embodiments, the kit further comprises auxiliary components for rebatch culture. In some embodiments, the kit further comprises auxiliary components for perfusion culture. In some embodiments, the auxiliary component is a hormone and / or other growth factor, inorganic ion, buffer, vitamin, nucleoside or nucleotide, trace element, amino acid, lipid, glucose or other energy source, and combinations thereof Selected from the group consisting of

  In this application, the use of “or” means “and / or” unless stated otherwise. As used in this application, the terms “comprise” and variations of this term such as “comprising” and “comprises” exclude other additives, components, integers or steps. Not intended. As used herein, the terms “about” and “approximate” are used as equivalents. All numbers used in this application with or without about (approximately / approximately) are meant to cover any normal variation understood by those skilled in the relevant art. In certain embodiments, the terms “about” or “about” mean that unless otherwise stated or otherwise obvious from the contents (such values are possible). 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, in both directions (greater or smaller) than the stated reference value) A range of values within 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less Say.

  Other features, objects, and advantages of the invention will be apparent from the following detailed description, drawings, and claims. However, it should be understood that the detailed description, drawings, and claims, while indicating embodiments of the invention, are given by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art.

  The drawings are for illustrative purposes only and are not intended to be limiting.

FIG. 6 is exemplary data illustrating the effect of various doses of iron addition after day 3 on cell growth of CHO cells in shake flasks. Cell growth is measured in terms of viable cell density (VCD) at a given time (total number of viable cells × 10 ⁶ cells / ml). FIG. 6 is exemplary data illustrating the effect of various doses of iron addition after day 3 on the viability of CHO cells in shake flasks. Viability is measured as a percentage of (viable cell count) / (total cell count) in culture at a given time point. FIG. 4 is exemplary data illustrating the effect of various doses of iron addition after day 3 on CHO cell viability in day 16 shake flasks. Viability is measured as a percentage of (viable cell count) / (total cell count) in culture at a given time point. FIG. 4 is exemplary data illustrating the effect of various doses of iron addition after day 3 on the antibody titer on day 16 of CHO cells in shake flasks. Antibody titer is measured in grams of antibody / liter culture medium. The change in antibody titer is measured as the ratio of [antibody] with _iron / [antibody] _{without iron} at a given time. 7 is exemplary data illustrating the improvement over cell density, viability, and titer control at day 14 when 1 mM iron is added to the cell line 2 culture process. Cell density is measured as the total number of viable cells at a given time point × 10 ⁶ cells / ml. The improvement in cell density _is expressed as the ratio of cell density _iron / _no cell density _iron . Viability is measured as a percentage of (viable cell count) / (total cell count) in culture at a given time point. Improvement in viability is measured as the ratio of _no% viability _{iron There} /% viability _iron. Antibody titer is measured in grams of antibody / liter culture medium. The change in antibody titer is measured as the ratio of [antibody] with _iron / [antibody] _{without iron} at a given time. Exemplary illustrating the effect of FeSO ₄ or Fe citrate addition (600 μM) after day 3 on viability and viable cell density of cell line 1 fed-batch culture in shake flasks on day 14 It is data. Viable cell density is measured as the total number of viable cells at a given time point × 10 ⁶ cells / ml. Improvement of viable cell density, expressed as the ratio of _no cell density _{iron There} / cell density _iron. 6 is exemplary data illustrating the effect of FeSO ₄ addition (1 mM) at day 6 on cell viability of CHO cells producing Nanobodies grown in fed-batch culture in shake flasks. 6 is exemplary data illustrating the effect of FeSO ₄ addition (1 mM) on day 6 on the titer of CHO cells producing Nanobodies grown in fed-batch culture in shake flasks. 6 is exemplary data illustrating the effect of FeSO ₄ addition (1 mM) at day 6 on the overall productivity of CHO cells producing Nanobodies grown in fed-batch culture in shake flasks. . 6 is exemplary data illustrating the effect of FeSO ₄ addition (1 mM) on day 6 on lactic acid production by CHO cells producing Nanobodies grown in fed-batch culture in shake flasks. FIG. 4 is exemplary data illustrating the effect of FeSO ₄ or Fe citrate addition (600 μM) on the cell density of fed-batch cultures in shake flasks of clone 2.8. FIG. 4 is exemplary data illustrating the effect of FeSO ₄ or Fe citrate addition (600 μM) on the viability of fed-batch cultures in shake flasks of clone 2.8. FIG. 4 is exemplary data illustrating the effect of FeSO ₄ or Fe citrate addition (600 μM) on the overall productivity of fed-batch cultures in shake flasks of clone 2.8. FIG. 3 is exemplary data illustrating the effect of various doses of iron addition at day 0 on the viability of cell line 1 at day 3 in shake flasks. Viability is measured as a percentage of (viable cell count) / (total cell count) in culture at a given time point.

  The present invention provides, among other things, methods and compositions that increase cell density, viability, and / or titer of a product by adding a composition comprising iron at one or more time points over the duration of the cell culture process. I will provide a. The present invention encompasses the surprising discovery that adding cell iron to the cell culture can delay and / or prevent cell death at the end of the cell culture. In some embodiments, iron can be added to the cell culture to inhibit apoptosis in the cell culture.

  Iron is a component of cell culture media that is important for the growth of mammalian cells. However, prior to the present invention, low levels of iron can inhibit cell growth when iron is depleted, while high levels of iron cause cell growth due to toxicity (eg, oxidative stress and free radical formation). It is thought that it can inhibit. Therefore, the balance between the beneficial and toxic properties of iron was thought to be important for mammalian cell culture. Prior to the present invention, conventional media formulations use low concentrations of iron to fully support cell growth while keeping below toxic iron concentration levels. As described in the Examples section, we have added iron, including high concentrations of iron, to the cell culture to improve cell growth and delay cell death in the cell culture. Was unexpectedly found to result in significantly increased cell density, viability and titer, among other benefits. Such effects are independent of cell line, scale, product and Fe source. Furthermore, the product quality was not affected by the high Fe concentration. Thus, the present invention provides a new and cost-effective approach for improved cell culture.

  Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can be applied to any aspect of the invention. However, one of ordinary skill in the art appreciates that various modifications to these embodiments are within the scope of the claims appended hereto. The scope of the present invention is defined by the claims and their equivalents, and the scope of the present invention is not limited by, and should not be limited to the specific embodiments or descriptions thereof.

Iron Compositions Various iron-containing compositions can be used according to the present invention. In certain embodiments, the composition comprising iron, FeSO _4, citric acid Fe, Fe- transferrin, chloride Fe, nitrate Fe, _Fe-EDTA, Fe a _{(NO 3) 3, FeCl 2} , FeCl 3 , and combinations thereof Selected from the group comprising

Various concentrations of iron can be used in accordance with the present invention. Typically, iron is provided in the medium at a concentration higher than the “minor concentration”. As used herein, the term “minor concentration” refers to a concentration that can be below normal or easily measured levels. For example, the trace level of the compound can be <10 ⁻⁵ , <10 ⁻⁶ , <10 ⁻⁷ or <10 ⁻⁸ M. In certain embodiments, the iron is at a concentration of about 100 μM to 5 mM (eg, about 100 μM to 4.5 mM, 100 μM to 3.0 mM, 100 μM to 2.5 mM, 100 μM to 1.0 mM, 100 μM to 1.0 mM, 200 μM). -2.0 mM, 200 μM-1.5 mM, 200 μM-1.0 mM, 150 μM-4.5 mM, 150 μM-3.5 mM, 150 μM-2.5 mM, 150 μM-1.5 mM, 150 μM-1.0 mM, 200 μM-1 5 mM, 200 μM to 1.0 mM, 200 μM to 2.5 mM, 300 μM to 2.5 mM, 300 μM to 1.5 mM, 300 μM to 2.0 mM). In certain embodiments, the iron is about 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900 μM, 950 μM, 1 mM, 1. Provided in the medium at concentrations of 5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any range within these concentrations. In certain embodiments, iron is provided in the medium at a concentration between about 300 μM and 1 mM. In some embodiments, the concentration of iron in the cell culture medium is increased over the duration of the cell culture process.

  The present invention also encompasses the discovery that a composition comprising iron can be provided in the medium at any point during the cell culture process. A composition comprising iron may be added by adding the composition at one or more time points over a period of time to achieve the desired iron concentration in the culture medium. In some embodiments, the composition comprising iron is added on or after day 0 of the cell culture process. In some embodiments, the composition comprising iron is added after day 0. In certain embodiments, the composition comprising iron is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, cell culture process (eg, production cell culture process). Add on day 13, 14, 15, 16, 17, 18, 19, 20 or later, or any combination of the above time points. In some embodiments, the composition comprising iron is added on or after day 3.

  Those skilled in the art will know the characteristics of the cells to be cultured, the characteristics of any product (eg, antibody or recombinant protein) produced by the cells in culture, the presence or absence of other components in the medium in which the cells are grown, Based on the particular attributes of the experimental design, including growth conditions, and the like, it would be possible to select the exact iron concentration and addition time within the scope of these inventions. For example, cells grown in static culture can utilize iron somewhat more optimally than cells grown in agitation culture (see, for example, WO 94/02592).

Cell Culture Medium As used herein, the terms “medium”, “cell culture medium” and “culture medium” refer to a solution containing nutrients that nourish growing mammalian cells. Typically, such solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. Such solutions also include, but are not limited to, hormones and / or other growth factors, certain ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace amounts Growth and / or including elements (usually inorganic compounds present at very low final concentrations), inorganic compounds present at high final concentrations (eg iron), amino acids, lipids, and / or glucose or other energy sources Auxiliary components that enhance survival beyond a minimal percentage may also be included. In certain embodiments, the medium is advantageously formulated at a pH and salt concentration that is optimal for cell survival and growth. In certain embodiments, the medium is a fed-batch medium that is added after the start of cell culture.

  A variety of mammalian growth media can be used in accordance with the present invention. In certain embodiments, the cells can be grown in one of a variety of known composition media where the components of the media are known and controlled. In certain embodiments, the cells can be grown in complex media where not all components of the media are known and / or controlled.

  Known composition growth media for mammalian cell culture have been developed and published on a large scale over the past decades. All components of the defined medium are well characterized and therefore the known medium does not contain complex additives such as serum or hydrolysates. Early media formulations were developed to allow maintenance of cell growth and viability with little or no concern for protein production. More recently, media formulations have been developed with the express purpose of supporting highly productive recombinant protein producing cell cultures.

  Not all components of complex media are well characterized, and therefore complex media contains, inter alia, simple and / or complex carbon sources, simple and / or complex nitrogen sources, and additives such as serum. Can do. In some embodiments, complex media suitable for the present invention contain additives such as hydrolysates in addition to other components of the known media described herein.

  In some embodiments, the known medium typically includes approximately 50 chemicals in water at known concentrations. Most of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin or BSA, while others do not require any protein components and are therefore known as protein-free known media. be called. The typical chemical components of the medium are divided into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and miscellaneous classifications that do not follow an organized classification.

  The cell culture medium may be supplemented with auxiliary components. The term “auxiliary component” as used herein includes, but is not limited to, hormones and / or other growth factors, certain ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffering agents. Minimal growth and / or survival, including vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources A component that enhances beyond proportion. In certain embodiments, auxiliary components can be added to the initial cell culture. In certain embodiments, auxiliary components may be added after the start of cell culture.

Typically, trace elements refer to various inorganic salts that are included at submicromolar levels. For example, generally contained trace elements are zinc, selenium, copper and the like. In some embodiments, iron (a divalent iron or trivalent iron salt) can be included as a trace element in micromolar concentrations in the initial cell culture medium. As described above, the present invention increases the iron concentration in the cell culture medium above a trace (eg, greater than 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1 mM). Thus, there is provided a method comprising adding additional iron to the trace amount of iron present in the initial cell culture medium. Manganese is also often included as a divalent cation (MnCl ₂ or MnSO ₄ ) in trace elements in the nanomolar to micromolar range. A number of less common trace elements are usually added at nanomolar concentrations.

Cells Any host cell that is amenable to cell culture can be utilized in accordance with the present invention. In certain embodiments, the host cell is a mammal. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include BALB / c mouse myeloma line (NSO / l, ECACC # 85110503), human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands), Monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney line (293 or 293 cells subcloned in suspension culture for growth, Graham et al., J. Gen Virol., 36:59, 1977), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells +/- DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216, 1980), Mouse Sertoli cells TM4, Mother, Biol.Reprod., 23: 243-251, 1980), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1 587), human cervical cancer cells (HeLa) ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse Breast cancer (MMT060562, ATCC CCL51), TRI cells (Mather et al., Anals NY Acad. Sci., 383: 44-68, 1982), MRC5 cells, FS4 cells, and human hepatocellular carcinoma line (Hep G2) Murrell.

  In addition, any number of commercially available and non-commercial hybridoma cell lines may be utilized in accordance with the present invention. As used herein, the term “hybridoma” refers to a cell or progeny of a cell resulting from the fusion of an immortalized cell and an antibody producing cell. The resulting hybridoma is an immortalized cell that produces antibodies. The individual cells used to make the hybridoma can be from any mammalian source including, but not limited to, rat, pig, rabbit, sheep, goat, and human. In certain embodiments, the hybridoma is a trioma cell that results when a progeny of a heterogeneous hybrid myeloma fusion that is the product of a fusion between a human cell and a murine myeloma cell line is subsequently fused with a plasma cell. It is a system. In certain embodiments, the hybridoma is any immortalized hybrid cell line that produces antibodies, such as, for example, quadromas (see, eg, Milstein et al., Nature, 537: 3053, 1983). One skilled in the art will understand that hybridoma cell lines may have different nutritional requirements and / or may require different culture conditions for optimal growth and can modify conditions as needed.

Cell Culture Methods As used herein, the terms “culture” and “cell culture” refer to a cell population suspended in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to those skilled in the art, in certain embodiments, these terms as used herein refer to a combination comprising a cell population and a medium in which the population is suspended. In certain embodiments, the cells of the cell culture comprise mammalian cells.

  The present invention may be used with any cell culture method that is amenable to the desired process (eg, production of recombinant proteins (eg, antibodies)). As a non-limiting example, the cells may be grown in batch or fed-batch culture, in which case the culture is stopped after the recombinant protein (eg, antibody) is fully expressed, and then the expressed protein ( Harvest for example antibodies). Alternatively, as another non-limiting example, the cells may be grown in rebatch or perfusion culture, in which case new nutrients and other components are cultured periodically or continuously without stopping the culture. In addition, recombinant proteins (eg, antibodies) expressed in the meantime are harvested regularly or continuously. Other suitable methods (eg, spin tube culture) are known in the art and can be used to practice the present invention.

  In certain embodiments, the cell culture suitable for the present invention is a fed-batch culture. As used herein, the term “fed-batch culture” refers to a cell culture method in which additional components are provided to the culture at one or more times following the start of the culture process. Such provided components typically include nutritional components for the cells that are depleted during the culturing process. In addition or alternatively, such additional components may include auxiliary components as described herein. In certain embodiments, the additional components are provided in a fed-batch medium as described herein. The fed-batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

  In certain embodiments, a cell culture suitable for the present invention is a rebatch culture. As used herein, the term “rebatch culture” refers to a portion of a cell (eg, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% of the cell after inoculation). , About 80%, about 85%, about 90%, about 95%, or more) are removed from the cell culture for a specific period of time (eg, 1 day, 2 days, 3 days, 4 days, 5 days, (6 days, 7 days, etc.) A cell culture method for growth. Typically, the cell-containing medium removed from the rebatch culture is replaced with an equal volume of fresh medium. In certain embodiments, the additional components are provided in the refeed media described herein. Re-batch culture is typically a continuous process that involves the expansion and maintenance of cell cultures in culture vessels (eg, bioreactors).

  In certain embodiments, a cell culture suitable for the present invention is a perfusion culture. Typically, perfusion culture involves the maintenance of a working volume of cell culture medium by the continuous introduction of fresh culture medium and the removal of spent medium through the use of a cell retention system. In some embodiments, the cells can be kept in culture using any available method, such as filtration, sedimentation, centrifugation, and combinations thereof. In some embodiments, the perfusion culture can be grown for an extended period (eg, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer).

  The cells can be grown in any convenient volume selected by the practitioner. For example, the cells can be grown in small scale reactors in a volume range from few milliliters to several liters. Alternatively, the cells are commercially available in a volume range of at least about 1 liter to 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000 liters or more, or any volume therebetween. In large scale bioreactors.

  The temperature of the cell culture is selected primarily based on the temperature range in which the cell culture remains viable and the range in which high levels of the desired product (eg, recombinant protein or antibody) are produced. For example, cell line 1 can grow well at about 37 ° C. and produce high titer antibodies. In general, most mammalian cells can grow well in the range of about 25 ° C. to 42 ° C. and produce the desired product (eg, recombinant protein or antibody), but the methods taught by this disclosure Is not limited to these temperatures. Certain mammalian cells can grow well in the range of about 35 ° C. to 40 ° C. and produce the desired product (eg, recombinant protein or antibody). In certain embodiments, the cell culture is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, at one or more times during the cell culture process. Growing at a temperature of 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ° C. One skilled in the art will be able to select the appropriate temperature or temperatures at which to grow the cells, depending on the specific requirements of the cells and the specific production requirements of the practitioner. The cells can be grown for any period of time, depending on the practitioner's requirements and the requirements of the cells themselves.

  The culture may be subjected to one or more temperature shifts over the duration of the culture. When shifting the temperature of the culture, the temperature shift can be relatively gradual. For example, it can take hours or days to complete the temperature change. Alternatively, the temperature shift can be relatively abrupt. The temperature can be gradually increased or decreased during the culturing process. Alternatively, the temperature can be increased or decreased in discrete amounts at various times during the culture process. The subsequent temperature (or temperatures) or temperature range (or temperature ranges) may be lower or higher than the initial or previous temperature (or temperatures) or temperature range (or temperature ranges) . One skilled in the art will appreciate that multiple discrete temperature shifts are encompassed within this embodiment. For example, the temperature may be shifted once (either to a higher or lower temperature or temperature range) and the cells are maintained at this temperature or temperature range for a period of time, after which the temperature is changed to the previous temperature or temperature range. The temperature may be shifted again to a new temperature or temperature range that may be either higher or lower than the temperature or temperature range. The temperature of the culture after each discrete shift can be constant or can be maintained within a specific temperature range.

  In certain embodiments, the cell density, viability, and / or titer of the cell culture is determined. As used herein, the term “cell density” refers to the number of cells present in a given volume of medium. The term “cell viability” as used herein refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to the portion of cells that are alive at that time in relation to the total number of viable and dead cells in the culture at a particular time. One skilled in the art will appreciate that one of many methods for determining cell viability is encompassed by the present invention. For example, to determine cell viability, a dye (eg, trypan blue) that does not pass through the membrane of live cells but can pass through the destroyed membrane of dead or dying cells may be used. Cell viability of cultures subjected to various culture conditions (eg, iron concentration or culture period) can be determined and compared to each other to determine optimal growth conditions for such cultures. As used herein, the term “titer” refers to the total amount of recombinantly expressed protein or antibody produced by, for example, a mammalian cell culture in a given volume of media. Titers are typically expressed in units of milligrams of protein, eg, antibody, per milliliter of medium.

  In certain embodiments, batch and fed-batch reactions are stopped when a desired cell density, viability, and / or titer is determined as determined by the practitioner's requirements. As a non-limiting example, the cells have a maximum viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, The culture can be stopped when it reaches 90, 95 or 99 percent. In addition or alternatively, batch and fed-batch reactions can be stopped before metabolic waste products such as lactic acid and ammonium accumulate excessively. In some embodiments, batch and fed-batch reactions are performed where waste accumulation (eg, lactic acid and / or ammonium) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / L culture medium. Or you can stop it when it gets higher.

  In certain cases, it may be beneficial to supplement the cell culture with nutrients or other media components that have been depleted or metabolized by the cells during the subsequent production phase. Non-limiting examples include hormones and / or other growth factors, inorganic ions (eg, sodium, chloride, calcium, magnesium, and phosphates), buffers, vitamins, nucleosides or nucleotides, trace elements, trace concentrations It may be beneficial to supplement the cell culture with high levels of inorganic compounds (eg iron), amino acids, lipids, or glucose or other energy sources. Such auxiliary components may be added to the cell culture at once, or they may be provided to the cell culture in a series of additions.

  Those skilled in the art will include, but are not limited to, growth rate, cell viability, final cell density of cell culture, final concentrations of harmful metabolic byproducts such as lactate and ammonium, desired products (eg, recombinant proteins or antibodies) Specific cell culture conditions to optimize specific characteristics of the cell culture, including any final titer, or any combination of these, or other conditions deemed important by the practitioner Will.

Protein Expression As noted above, in many cases, cells are selected or manipulated to produce high levels of the desired product (eg, recombinant protein or antibody). Often, in order to produce high levels of recombinant protein, for example, by introducing a gene encoding the protein of interest and / or expressing the gene (whether endogenous or introduced). Cells are manipulated by human hands by the introduction of regulatory genes that regulate them.

  Certain proteins can have detrimental effects on cell growth, cell viability, or some other characteristic of the cell that ultimately limits the production of the protein of interest in some manner. A protein of interest where even within a particular population of cells that are engineered to express a particular protein, variation within the cell population exists to allow a particular individual cell to grow better Or a protein with a higher activity level (eg, enzyme activity). In certain embodiments, the cell line is empirically selected by the practitioner for robust growth under the specific conditions selected to culture the cells. In some embodiments, individual cells engineered to express a particular protein are cell growth, final cell density, percent cell viability, expressed protein titer or any combination thereof, or Selected for large scale production based on any other conditions deemed important by the practitioner.

  Any protein that can be expressed in a host cell can be produced according to the present teachings. As used herein, the term “host cell” refers to a cell that has been engineered according to the present invention to produce a protein of interest as described herein. In some embodiments, the host cell is a mammalian cell. Proteins can be expressed from genes that are endogenous to the host cell or from heterologous genes that have been introduced into the host cell. The protein can be naturally occurring or have a sequence that has been manipulated or selected by the hand of man.

  Proteins that can be desirably expressed according to the present invention are often selected based on interesting or useful biological or chemical activity. For example, the present invention can be used to express any pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone, modulator, antigen, binding agent, and the like. In some embodiments, the protein expressed by the cells in culture is an antibody or fragment thereof, Nanobody, single domain antibody, glycoprotein, therapeutic protein, growth factor, clotting factor, cytokine, fusion protein, drug substance , Vaccine, enzyme, or Small Modular ImmunoPharmaceutical ™ (SMIP). The list of proteins that can be produced according to the present invention is merely exemplary in nature and is not intended to be a limiting listing. Those skilled in the art will understand that any protein can be expressed in accordance with the present invention, and will be able to select the particular protein to be produced based on their particular needs.

Antibodies In view of the large number of antibodies currently used or under investigation as pharmaceuticals or other commercially available drugs, the production of antibodies is of particular interest according to the present invention. An antibody is a protein that has the ability to specifically bind to a particular antigen. Any antibody that can be expressed in a host cell can be produced according to the present invention. In some embodiments, the antibody to be expressed is a monoclonal antibody.

  In some embodiments, the monoclonal antibody is a chimeric antibody. A chimeric antibody contains amino acid fragments derived from multiple organisms. A chimeric antibody molecule can include, for example, an antigen binding domain from a mouse, rat, or other species of antibody along with a human constant region. Various techniques for making chimeric antibodies have been described. For example, Morrison et al., Proc. Natl. Acad. Sci. U. S. A. 81, 6851 (1985), Takeda et al., Nature, 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567, Boss et al., U.S. Pat. No. 4,816,397, Tanaguchi et al., Europe. See patent publication EP 17196, European patent publication 0173494, British patent GB 2177096B.

  In some embodiments, monoclonal antibodies are used, for example, in the use of ribosome display or phage display libraries (see, eg, Winter et al., US Pat. No. 6,291,159 and Kawasaki, US Pat. No. 5,658,754). Or the use of xenografts that inactivate the native antibody gene and functionally replace it with a human antibody gene, while leaving other components of the native immune system intact (eg Kucherlapati et al., US Pat. No. 6, 657,103)).

  In some embodiments, the monoclonal antibody is a humanized antibody. A humanized antibody is a chimeric antibody in which the majority of amino acid residues are derived from a human antibody, thus minimizing all potential immune responses when delivered to a human subject. In humanized antibodies, amino acid residues in the complementarity determining regions are at least partially replaced with residues from non-human species that confer the desired antigen specificity or affinity. Such altered immunoglobulin molecules can be made by any of a number of techniques known in the art (see, eg, Teng et al., Proc. Natl. Acad. Sci. U.S.). S.A., 80, 7308-7312 (1983), Kozbor et al., Immunology Today, 4, 7279 (1983), Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), preferably all PCT Publication No. WO 92/06193 or EP 0239400, which is incorporated herein by reference). Humanized antibodies can be produced commercially, for example, by Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, UK. Additional references include Jones et al., Nature, 321: 522-525 (1986), Riechmann et al., Nature, 332: 323-329 (1988), and Presta, Curr., All incorporated herein by reference. Op. Struct. Biol. 2: 593-596 (1992).

  In some embodiments, the monoclonal, chimeric, or humanized antibody described above may contain amino acid residues that are not naturally found in any antibody of any species in nature. These foreign residues can be utilized, for example, to confer new or modified specificity, affinity or effector function to monoclonal, chimeric or humanized antibodies. In some embodiments, the antibodies described above can be conjugated to drugs for systemic drug therapy, such as toxins, low molecular weight cytotoxic drugs, biological response modifiers, and radionuclides (eg, Kunz). Et al., Calicheamic derivative-carrier conjugates, US20040082764 A1).

  In some embodiments, the present invention is used to accumulate amyloid lytic plaques in the brain that specifically bind to Aβ fragments of amyloid precursor protein or other components of amyloid lytic plaques and characterize Alzheimer's disease. Produce useful antibodies to fight. (See, eg, US Provisional Application 60 / 636,684). In some embodiments, the present invention is used to produce antibodies that specifically bind to the HER2 / neu receptor. In some embodiments, the present invention is used to produce anti-CD20 antibodies. In some embodiments, the invention is used to produce antibodies to TNFα, CD52, CD25, VEGF, EGFR, CD11a, CD33, CD3, IL-22, alpha-4 integrin, and / or IgE.

  In another embodiment, the antibodies of the invention are directed against cell surface antigens expressed on target cells and / or tissues within proliferative disorders such as cancer. In one embodiment, the antibody is an IgG1 anti-Lewis Y antibody. Lewis Y is a carbohydrate antigen having the structure Fucα1 → 2Galβ1 → 4 [Fucα1 → 3] GlcNacβ1 → 3R (Abe et al. (1983) J. Biol. Chem. 258, 11793-11797). Lewis Y antigen is expressed on the surface of 60% -90% of human epithelial tumors (including those of breast, colon, lung, and prostate), at least 40% of which overexpress this antigen, and in normal tissues Expression is restricted.

  In order to target Ley and effectively target the tumor, an antibody with exclusive specificity for the antigen is ideally needed. Thus, preferably, the anti-Lewis Y antibodies of the invention do not cross-react with type 1 structures (ie blood group lacto series (Lea and Leb)), preferably other 2 types such as Lex and H-2 type structures. Does not bind to type epitope (ie neolacto structure). An example of a preferred anti-Lewis Y antibody is designated hu3S193 (US Pat. Nos. 6,310,185, 6,518,415, 5,874, which are incorporated herein in their entirety). (See 060). The humanized antibody hu3S193 (Attia, MA, et al., 1787-1800) was obtained by CDR grafting from 3S193, a murine monoclonal antibody raised against adenocarcinoma cells with exceptional specificity for Ley. (Kitamura, K., 12957-12961). hu3S193 not only retains the specificity of 3S193 for Ley, but also complement-dependent cytotoxicity (hereinafter referred to as CDC) and antibody-dependent cellular cytotoxicity (hereinafter referred to herein as). Has also acquired the ability to mediate (referred to as ADCC) (Attia, MA, et al., 1787-1800). This antibody targets xenografts expressing Ley in nude mice, which require hu3S193 labeled with 125I, 111In, or 18F, and other chelating agents such as 111In, 99mTc, or 90Y Has been demonstrated by biodistribution studies using the following radiolabels (Clark et al., 4804-4811).

  In some embodiments, the antibody is one of the human anti-GDF-8 antibodies designated Myo29, Myo28, and Myo22, and antibodies and antigen-binding fragments derived therefrom. These antibodies can bind mature GDF-8 with high affinity and inhibit GDF-8 activity in vitro and in vivo, as demonstrated, for example, by inhibition of ActRIIB binding and reporter gene assays, and GDF-8 activity associated with negative regulation of skeletal muscle mass and bone density can be inhibited. See, for example, Veldman et al., U.S. Patent Application No. 200404142382.

Receptors Another polypeptide class that has been shown to be effective as pharmaceuticals and / or commercially available drugs includes receptors. Receptors are typically transmembrane glycoproteins that function by recognizing extracellular signaling ligands. Receptors typically have a protein kinase domain in addition to a ligand recognition domain that, when bound to a ligand, initiates a signal transduction pathway by phosphorylating a target intracellular molecule, which initiates cellular signaling. Cause developmental or metabolic changes in the body. In one embodiment, the receptor of interest is modified to remove the transmembrane and / or intracellular domain (or domains), and instead may have an Ig-domain attached. In a preferred embodiment, the receptor produced according to the present invention is a receptor tyrosine kinase (RTK). The RTK family includes receptors that are important for a variety of functions in a number of cell types (see, eg, Yarden and Ulrich, Ann. Rev. Biochem., 57: 433-incorporated herein by reference). 478, 1988, Ullrich and Schlessinger, Cell, 61: 243-254, 1990). Non-limiting examples of RTKs include members of the fibroblast growth factor (FGF) receptor family, epidermal growth factor receptor (EGF) family, platelet derived growth factor (PDGF) receptor, immunoglobulins and EGF Tyrosine kinases having homology domain-1 (TIE-1) and TIE-2 receptors (Sato et al., Nature, 376 (6535): 70-74 (1995), incorporated herein by reference), As well as c-Met receptors, some of which have been suggested to directly or indirectly promote angiogenesis (Mustonen and Alitaro, J. Cell Biol., 129: 895-898, 1995). Other non-limiting examples of RTKs include fetal liver kinase 1 (FLK-1) (kinase insert domain containing receptor (KDR) (Terman et al., Oncogene, 6: 1677-83, 1991) or vascular endothelial cell growth. Factor receptor 2 (sometimes referred to as VEGFR-2)), fms-like tyrosine kinase-1 (Flt-1) (DeVries et al., Science 255, 989-991, 1992, Shibuya et al., Oncogene, 5: 519- 524, 1990) (sometimes referred to as vascular endothelial growth factor receptor 1 (VEGFR-1)), neuropilin-1, endoglin, endosialin, and Axl. Those skilled in the art will recognize other receptors that can be preferably expressed in accordance with the present invention.

  In some embodiments, the tumor necrosis factor inhibitor is a tumor necrosis factor alpha and beta receptor (TNFR-1, EP 417,563 published 20 March 1991, and TNFR-2, 20 March 1991). Expressed in the form of Japanese published EP 417,014) (in the review, Naismith and Sprang, J Inflamm., 47 (1-2): 1: 1, incorporated herein by reference). 7 (1995-96)). According to one embodiment, the tumor necrosis factor inhibitor comprises a soluble TNF receptor and preferably TNFR-Ig. In some embodiments, preferred TNF inhibitors of the present invention are soluble forms of TNFRI and TNFRII and soluble TNF binding proteins. In another embodiment, the TNFR-Ig fusion is TNFR: Fc, and the term refers to “etanercept” as used herein. This is a dimer of two molecules of the extracellular portion of the p75TNF-α receptor, each consisting of a 235 amino acid Fc portion of human IgG1.

Growth Factors and Other Signaling Molecules Another polypeptide class that has been shown to be effective as pharmaceuticals and / or commercially available drugs includes growth factors and other signaling molecules. Growth factors include glycoproteins that are secreted by cells and bind to and activate receptors on other cells to initiate metabolic or developmental changes in the receptor cells. In one embodiment, the protein of interest is an ActRIIB fusion polypeptide comprising the extracellular domain of the ActRIIB receptor and the Fc portion of the antibody (see, eg, Wolfman et al., ActRIIB fusion polypeptides and uses thefore, US 2004/0223966 A1). . In another embodiment, the growth factor can be a modified GDF-8 propeptide (see, eg, Wolfman et al., Modified and stabilized GDF propeptides and uses thereof, US 2003/0104406 A1). Alternatively, the protein of interest can be a follistatin-domain containing protein (eg, Hill et al., GASP1: a follatin domain domaining protein, US2003 / 0162714 A1, Hill et al., GASP1: a follistin domain domain 10 5Atin 10 USA 10 5 No., Hill et al., Follistatin domain containing proteins, US 2003/0180306 A1).

  Non-limiting examples of mammalian growth factors and other signaling molecules include cytokines, epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factors (FGF) such as aFGF and bFGF, Transforming growth factors (TGF), such as TGF-alpha and TGF-beta, including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5, insulin-like growth factor- I and -II (IGF-I and IGF-II), Death (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein, CD-3, CD-4, CD-8, And CD proteins such as CD-19, erythropoietin, osteoinductive factor, immunotoxin, bone morphogenetic protein (BMP), inter Interferons such as ferron-alpha, -beta, and -gamma, colony stimulating factor (CSF), eg, M-CSF, GM-CSF, and G-CSF, interleukin (TL), eg, IL-1 to IL-10 , IL-10 superfamily cytokines (eg, IL-19, IL-20, IL-22, IL-24, IL-26), tumor necrosis factor (TNF) alpha and beta, insulin A chain, insulin B chain, pro Coagulation factors such as insulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, factor VIIIC, factor IX, tissue factor and von Willebrand factor, anticoagulant factors such as protein C, atrial natriuretic factor, lung Surfactant, urokinase or human urine or Plasminogen activators such as woven plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factor, enkephalinase, RANTES (regulated on activation, normal T-cell expressed and secreted), Human macrophage inflammatory protein (MIP-1-alpha), Muller tube inhibitor, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin related peptide, bone-derived neurotrophic factor (BDNF), neurotrophin-3, − Neurotrophic factors such as 4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6) or nerve growth factors such as NGF-beta are included. Those skilled in the art will recognize other growth factors or signaling molecules that can be expressed in accordance with the present invention.

G-protein coupled receptors Another polypeptide class that has been shown to be effective as pharmaceuticals and / or commercially available drugs includes growth factors and other signaling molecules. A G-protein coupled receptor (GPCR) is a protein having seven transmembrane domains. When the ligand and GPCR are combined, a signal is transmitted within the cell, resulting in a change in the biological or physiological properties of the cell.

  GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are components of a modular signaling system that links the state of intracellular second messengers with extracellular inputs. These genes and gene products are potential causative agents of the disease.

  Specific deficiencies in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of autosomal dominant and autosomal recessive retinitis pigmentosa, nephrogenic diabetes insipidus. These receptors are of critical importance in both the central nervous system and peripheral physiological processes. The GPCR protein superfamily currently contains over 250 paralogs (receptors that represent variants produced by gene duplication (or other processes) relative to orthologs that are the same receptor from different species). This superfamily can be divided into the following five families: Family I: Receptors typified by rhodopsin and beta2-adrenergic receptors, currently represented by over 200 unique members Family II: recently characterized parathyroid hormone / calcitonin / secretin receptor family, family III: metabotropic glutamate receptor family in mammals, family IV: cAMP receptor family, D. discoidideum ) And chemotactic and developmental and family V: fungal mating pheromone receptors such as STE2.

  GPCRs include receptors for biogenic amines, inflammatory lipid mediators, peptide hormones, and sensory signal mediators. A GPCR is activated when a receptor binds to its extracellular ligand. Conformational changes in the GPCR resulting from ligand-receptor interactions affect the binding affinity of the G protein for the GPCR intracellular domain. This allows GTP to bind to the G protein with enhanced affinity.

  Activation of the G protein by GTP results in the interaction of the G protein α subunit with adenylate cyclase or other second messenger molecule generators. This interaction regulates the activity of adenylate cyclase and thus the production of the second messenger molecule cAMP. cAMP regulates phosphorylation and activation of other intracellular proteins. Alternatively, cellular levels of other second messenger molecules such as cGMP or eicosanoids can be upregulated or downregulated by the activity of the GPCR. The G protein a subunit is inactivated by GTP hydrolysis by GTPase, and the α, β, and γ subunits reassociate. This heterotrimeric G protein then dissociates from adenylate cyclase or other second messenger molecule generators. GPCR activity can also be regulated by phosphorylation of intracellular and extracellular domains or loops.

  Glutamate receptors are an important group of GPCRs in neurotransmission. Glutamate is a major neurotransmitter in the CNS and is believed to have an important role in neuroplasticity, cognition, memory, learning and some neurological disorders such as epilepsy, stroke, and neurodegeneration (Watson, S And S. Arkinstal (1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego CA, pages 130-132). These effects of glutamate are mediated by two different receptor classes, termed ionic and metabolic. The ionotropic receptor contains an endogenous cation channel and mediates the fast excitatory action of glutamate. Metabotropic receptors are regulatory and increase neuronal membrane excitability by inhibiting calcium-dependent potassium conductance and inhibiting and enhancing excitatory transmission of ionotropic receptors. Metabolic receptors are classified into five subtypes based on agonist pharmacology and signaling pathways and are widely distributed in brain tissue.

  The vasoactive intestinal polypeptide (VIP) family is a group of related polypeptides whose actions are also mediated by GPCRs. The major members of this family are VIP itself, secretin, and growth hormone releasing factor (GRF). VIP has a broad physiological profile, including smooth muscle relaxation, stimulation or inhibition of secretion in various tissues, modulation of various immune cell activities, and various excitatory and inhibitory activities in the CNS. Secretin stimulates the secretion of enzymes and ions in the pancreas and intestinal tract and is also present in small amounts in the brain. GRF is an important neuroendocrine substance that regulates growth hormone synthesis and release from the anterior pituitary gland (Watson, S. and S. Arkinstal, supra, pages 278-283).

  After ligand binding to the GPCR, a conformational change is transmitted to the G protein, whereby the α-subunit exchanges the bound GDP molecule for GTP molecules and dissociates from the βγ-subunit. Is caused. The α-subunit in a form associated with GTP typically functions as an effector modulating moiety, and produces second messengers such as cyclic AMP (eg, by activation of adenylate cyclase), diacylglycerol or inositol phosphates. Bring. More than 20 different types of α-subunits are known in humans and these associate with a smaller pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. The G protein is a protein from Rhodis H. et al., The contents of which are incorporated herein by reference. Et al, Molecular Cell Biology, (Scientific American Books Inc., New York, NY, 1995).

  GPCRs are major targets for drug action and development. In fact, the receptors yield more than half of the currently known drugs (Drews, Nature Biotechnology, 1996, 14: 1516) and GPCRs represent the most important target of therapeutic action and are clinically prescribed drugs 30% either antagonize or stimulate the GPCR (Milligan, G. and Rees, S., (1999) TIPS, 20: 118-124). This demonstrates that these receptors have an established and proven history as therapeutic targets.

  In general, the practitioner of this invention will select the polypeptide of interest and know its exact amino acid sequence. Any given protein expressed in accordance with the present invention has its own idiosyncratic characteristics and may affect the cell density or viability of the cultured cells, and other proteins grown under the same culture conditions It may be expressed at a lower level than the polypeptide or protein. Those skilled in the art will be able to modify the steps and compositions of the invention appropriately to optimize cell growth and / or production of any given expressed polypeptide or protein.

Enzymes Another protein class that has been shown to be effective as pharmaceuticals and / or commercially available drugs includes enzymes. Enzymes can be proteins whose enzymatic activity can be affected by the cell culture conditions that produced them. Accordingly, it is also of particular interest to produce enzymes having the desired enzyme activity according to the present invention. Those skilled in the art will recognize a number of known enzymes that can be expressed by cells in culture.

  Non-limiting examples of enzymes include amylases, cellulases, dextranases, glucosidases, galactosidases, glucoamylases, hemicellulases, pentosanases, xylanases, invertases, lactases, naringinases, pectinases, pullulanases and other carbohydrases, acidic proteases, alkaline proteases , Proteases such as bromelain, ficin, neutral protease, papain, pepsin, peptidase (eg, aminopeptidase and carboxypeptidase), rennet, rennin, chymosin, subtilisin, thermolysin, aspartic proteinase, and trypsin, triglyceridase, phospholipase, Pregastric esterase, phosphatase, phytase, amidase, iminoacyler Lipase or esterase such as glutaminase, lysozyme, and penicillin acylase, isomerase such as glucose isomerase, amino acid oxidase, catalase, chloroperoxidase, glucose oxidase, oxidoreductase such as hydroxysteroid dehydrogenase or peroxidase, acetolactate decarboxylase, asparagine Includes acid decarboxylase, elimination enzyme such as fumarase or histase, transferase such as cyclodextrin glycosyltransferase, ligase, chitinase, cutinase, deoxyribonuclease, laccase, mannosidase, mutanase, pectin degrading enzyme, polyphenol oxidase, ribonuclease and transglutaminase It is.

  In general, the practitioner of this invention will select the protein of interest and know its exact amino acid sequence. Any given protein expressed in accordance with the present invention may have its own particular characteristics, which may affect the cell density or viability of the cultured cells, and is different from those grown under the same culture conditions. It may be expressed at a lower level than the protein and may have different biological activities depending on the exact culture conditions and steps taken. Those skilled in the art will appropriately use the steps and compositions used to produce a particular protein in accordance with the teachings of the present invention to optimize cell growth and the level of production and / or activity of any given expressed protein. Could be modified.

Introduction of a gene for protein expression into a host cell In general, a nucleic acid molecule introduced into a cell encodes a protein that is desired to be expressed according to the present invention. Alternatively, the nucleic acid molecule can encode a gene product that induces expression of the desired protein by the cell. For example, the introduced genetic material can encode a transcription factor that activates transcription of endogenous or heterologous proteins. Alternatively or additionally, the introduced nucleic acid molecule can increase the translation or stability of the protein expressed by the cell.

  Suitable methods for introducing sufficient nucleic acid into a mammalian host cell to achieve expression of the protein of interest are known in the art. For example, Gething et al., Nature, 293: 620-625, 1981, Mantei et al., Nature, 281: 40-46, 1979, Levinson et al., EP 117,060, each of which is incorporated herein by reference. And EP 117,058. For mammalian cells, common methods for introducing genetic material into mammalian cells include Graham and van der Erb's calcium phosphate precipitation method (Virology, 52: 456-457, 1978) or Hawley-Nelson's lipofectamine (trademark). ) (Gibco BRL) method (Focus, 15:73, 1993). General aspects of transformation of mammalian cell host systems are described by Axel in US Pat. No. 4,399,216 issued Aug. 16, 1983. Various techniques for introducing genetic material into mammalian cells include Keown et al., Methods in Enzymology, 1989, Keown et al., Methods in Enzymology, 185: 527-537, 1990, and Mansour et al., Nature 336: 348-352, 1988.

  In some embodiments, the introduced nucleic acid is in the form of a naked nucleic acid molecule. For example, a nucleic acid molecule introduced into a cell can consist only of the nucleic acid encoding the protein and the necessary gene regulators. Alternatively, a nucleic acid encoding the protein (including necessary regulatory elements) can be included in the plasmid vector. Non-limiting representative examples of suitable vectors for expressing proteins in mammalian cells include pCDNA1, pCD (see Okayama et al., Mol. Cell Biol., 5: 1136-1142, 1985), pMClneo poly- A (see Thomas et al., Cell, 51: 503-512, 1987), baculoviral vectors such as pAC373 or pAC610, CDM8 (see Seed, B., Nature, 329: 840, 1987), and pMT2PC (Kaufman et al. , EMBO J., 6: 187-195, 1987), each of which is incorporated herein by reference in its entirety. In some embodiments, the nucleic acid molecule to be introduced into the cell is contained within a viral vector. For example, a nucleic acid encoding a protein can be inserted into the viral genome (or partial viral genome). The regulatory elements that direct the expression of the protein can be included with the nucleic acid inserted into the viral genome (ie linked to a gene inserted into the viral genome) or provided by the viral genome itself.

  Naked DNA can be introduced into cells by forming a precipitate containing DNA and calcium phosphate. Alternatively, by forming a mixture of DNA and DEAE-dextran and incubating the mixture with the cells or by incubating the cells and DNA together in an appropriate buffer and subjecting the cells to a high voltage electrical pulse ( Naked DNA can also be introduced into cells (eg, by electroporation). A further method for introducing naked DNA into cells is by mixing the DNA with a liposome suspension containing a cationic lipid. The DNA / liposome complex is then incubated with the cells. Alternatively, naked DNA can be directly injected into cells, for example, by microinjection.

  Alternatively, naked DNA can also be introduced into cells by complexing the DNA with a cation such as polylysine that is coupled to a ligand for cell surface receptors (eg, each of which is in its entirety). Wu, G. and Wu, CH, J. Biol. Chem., 263: 14621, 1988, Wilson et al., J. Biol. Chem., 267: 963, incorporated herein by reference. -967, 1992, and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates DNA uptake by receptor-mediated endocytosis.

  The use of viral vectors containing a specific nucleic acid sequence, eg, a cDNA encoding a protein, is a common technique for introducing nucleic acid sequences into cells. Infection of cells with viral vectors has the advantage that the majority of cells receive the nucleic acid, which can eliminate the need for selection of cells that have received the nucleic acid. Furthermore, molecules encoded within a viral vector, eg, by the cDNA contained in the viral vector, are generally efficiently expressed in cells that have taken up the viral vector nucleic acid.

  Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (see Miller, AD, Blood, 76: 271, 1990 for a review). A recombinant retrovirus can be constructed in which a nucleic acid encoding the protein of interest is inserted into the retroviral genome. In addition, a portion of the retroviral genome can be removed to render the retrovirus replication defective. Such replication-defective retroviruses can then be packaged into virions and used to infect target cells using helper viruses by standard techniques.

  The adenovirus genome encodes and expresses the protein of interest but can be engineered to be inactivated with respect to its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques, 6: 616, 1988, Rosenfeld et al., Science, 252: 431-434, 1991, and Rosenfeld et al., Cell, 68: 143-155, 1992. Appropriate adenoviral vectors derived from the adenovirus strain Ad5 type dl324 or other strains of adenovirus (eg, Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses do not require dividing cells to be effective gene delivery vehicles, and include airway epithelium (Rosenfeld et al., 1992, cited above), endothelial cells (Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89: 6482-6486, 1992), hepatocytes (Herz and Gerard, Proc. Natl. Acad. Sci. USA, 90: 2812-1816, 1993), and muscle cells (Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, 1992), which is advantageous in that it can be used to infect various cell types. Furthermore, the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the host cell genome, but remains episomal, thereby allowing the introduced DNA to be integrated into the host genome. Potential problems that may occur as a result of insertional mutations (eg retroviral DNA) are avoided. Furthermore, the adenoviral genome has a greater capacity for foreign DNA (up to 8 kilobases) compared to other gene delivery vectors (Berkner et al., Cited above, Haj-Ahmand and Graham, J. Virol., 57: 267, 1986). Most replication-deficient adenoviral vectors currently in use lack the viral E1 and E3 genes in whole or in part, but retain as much as 80% of the adenoviral genetic material.

  Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus as a helper virus, such as adenovirus or herpes virus, for efficient replication and a productive life cycle. (See Muzyczka et al., Curr. Topics in Micro. And Immunol., 158: 97-129, 1992 for a review). It is also one of the few viruses that can incorporate its DNA into non-dividing cells and exhibits a high frequency of stable integration (see, eg, Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7: 349-356, 1992, Samulski et al., J. Virol., 63: 3822-3828, 1989, and McLaughlin et al., J. Virol., 62: 1963-1973, 1989). Less than that, vectors containing 300 base pairs of AAV can be packaged and incorporated. Space for exogenous DNA is limited to about 4.5 kb. DNA can be introduced into cells using AAV vectors such as those described in Tratschin et al. (Mol. Cell. Biol., 5: 3251-3260, 1985). Various nucleic acids have been introduced into various cell types using AAV vectors (eg, Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466-6470, 1984, Tratschin et al., Mol. Cell. Biol., 4: 2072-2081, 1985, Wondisford et al., Mol. Endocrinol., 2: 32-39, 1988, Tratschin et al., J. Virol., 51: 611-619, 1984, and Flotte et al., J. Biol. Biol.Chem., 268: 3781-3790, 1993).

  If the method used to introduce the nucleic acid molecule into the cell population results in the modification of most cells and efficient expression of the protein by the cells, the modified cell population is an additional unit of individual cells within the population. Can be used without isolation or subcloning. That is, there may be sufficient production of the protein by the cell population so that no further cell isolation is necessary, and the population can be used immediately to inoculate the cell culture for protein production. Alternatively, it may be desirable to isolate a group of cells or a single cell that efficiently produces the protein and then expand the uniform cell population.

  Instead of introducing a nucleic acid molecule encoding the protein of interest into the cell, the introduced nucleic acid induces or increases the level of a protein produced endogenously by the cell, or another polypeptide or It may encode a protein. For example, a cell may express a particular protein but may not express without additional processing of the cell. Similarly, cells may express insufficient amounts of protein for the desired purpose. Thus, an agent that stimulates expression of a protein of interest can be used to induce or increase expression of that protein by the cell. For example, the introduced nucleic acid molecule may encode a transcription factor that activates or upregulates transcription of the protein of interest. The expression of such a transcription factor thus leads to the expression of the target protein, or more robust expression.

  In certain embodiments, nucleic acids that direct protein expression are stably introduced into host cells. In certain embodiments, a nucleic acid that directs expression of the protein is transiently introduced into the host cell. One skilled in the art can choose whether to introduce the nucleic acid stably or transiently into the cell based on their experimental requirements.

  The gene encoding the protein of interest may be linked to one or more regulatory gene regulators. In certain embodiments, the gene regulator directs constitutive expression of the protein. In certain embodiments, a genetic regulator that results in inducible expression of a gene encoding the protein of interest can be used. The use of inducible gene regulators (eg inducible promoters) allows modulation of protein production in the cell. Non-limiting examples of inducible gene regulators that are potentially useful for use in eukaryotic cells include hormone regulatory factors (eg, Mader, S. and White, JH, Proc. Natl. Acad). USA, 90: 5603-5607, 1993), synthetic ligand modulators (see, eg, Spencer, DM et al., Science, 262: 1019-1024, 1993), and ionizing radiation modulators (see For example, see Manome, Y. et al., Biochemistry, 32: 10607-10613, 1993, Datta, R. et al., Proc. Natl. Acad. Sci. USA, 89: 10149-10153, 1992). Additional cell specific or other regulatory systems known in the art may be used in accordance with the present invention.

  Those skilled in the art will be able to select and optionally modify appropriately the method of introducing the gene that causes the cell to express the protein of interest in accordance with the teachings of the present invention.

Isolation of Expressed Protein In general, it will typically be desirable to isolate and / or purify proteins expressed in accordance with the present invention. In certain embodiments, the expressed protein is secreted into the medium, and thus cells and other solids can be removed, for example, by centrifugation or filtration as the first step in the purification process.

  Alternatively, the expressed protein may be bound to the surface of the host cell. For example, media can be removed and host cells expressing the protein can be lysed as the first step in the purification process. Lysis of mammalian host cells can be achieved by any number of means well known to those skilled in the art, including physical disruption with glass beads and exposure to high pH conditions.

  Expressed proteins include but are not limited to chromatography (eg, ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation, and / or protein. Can be isolated and purified by standard methods, including by any other available techniques for purifying (eg, Scopes, Protein Purification Principles, each of which is incorporated herein by reference) and Practice, 2nd edition, Springer-Verlag, New York, 1987, Higgins, SJ and Hames, BD (ed.), Protein Expressio. : A Practical Approach, Oxford Uniform Press, 1999, and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods 182), Academic Press, 1997). Particularly in immunoaffinity chromatography, the protein can be isolated by binding to an affinity column that contains the antibody immobilized on a fixed support that is produced against the protein. Alternatively, an affinity tag such as an influenza coat sequence, poly-histidine, or glutathione-S-transferase is attached to the protein by standard recombinant techniques to allow easy purification by passing through an appropriate affinity column. Can do. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be added at any or all stages to reduce or eliminate protein degradation during the purification process. Protease inhibitors are particularly advantageous when cells must be lysed to isolate and purify the expressed protein.

  One skilled in the art will appreciate that the exact purification technique will vary depending on the characteristics of the protein to be purified, the characteristics of the cells in which the protein is expressed, and / or the composition of the medium in which the cells are grown.

Pharmaceutical Formulations In certain preferred embodiments of the invention, the produced polypeptide or protein has pharmacological activity and is useful in the preparation of a pharmaceutical product. The compositions of the present invention described above can be administered to a subject, or initially, but not exclusively, parenteral (eg, intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (Topical), transmucosal, rectal, and vaginal routes may be formulated for delivery by any available route. The pharmaceutical compositions of the invention typically include purified polypeptides or proteins expressed from mammalian cell lines, delivery agents (ie, cationic polymers, peptide molecular transporters, surfactants as described above). Etc.) in combination with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Is included. Supplementary active compounds can also be incorporated into the compositions.

  A pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: water for injection, saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or Aseptic diluents such as other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, acetates, citrates or phosphates Buffers and agents that adjust isotonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Is included. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. Preferred pharmaceutical formulations must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. In general, the associated carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

  A sterile injectable solution is prepared by incorporating the purified polypeptide or protein in the required amount, optionally in one or a combination of the above-listed components, in a suitable solvent and then sterile filtered. be able to. In general, dispersions are prepared by incorporating purified polypeptide or protein expressed from a mammalian cell line into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. To do. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, whereby any additional ingredients from the active ingredient and its previously sterile filtered solution are obtained. A powder of the desired component is obtained.

  Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the purified polypeptide or protein can be incorporated with excipients and used in the form of tablets, troches, or capsules, eg, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar properties: binders such as crystalline cellulose, gum tragacanth or gelatin, excipients such as starch or lactose Agents, alginic acids, Primojel® or disintegrants such as corn starch, lubricants such as magnesium stearate or Sterotes, glidants such as colloidal silicon dioxide, sweeteners such as sucrose or saccharin, or peppermint, methyl salicylate Or flavoring such as orange flavor. Formulations for oral delivery may advantageously incorporate agents that improve stability in the gastrointestinal tract and / or enhance absorption.

  For administration by inhalation, a composition of the invention comprising a purified polypeptide or protein expressed from a mammalian cell line and a delivery agent is preferably pressurized containing a suitable propellant, eg, a gas such as carbon dioxide. Delivered in the form of an aerosol spray from a container or dispenser, or nebulizer. The present invention specifically contemplates delivery of the composition using a nasal spray, inhaler, or other direct delivery to the upper and / or lower respiratory tract. Intranasal administration of a DNA vaccine directed against influenza virus has been shown to induce a CD8 T cell response, which means that at least some cells in the respiratory tract take up DNA when delivered by this route The delivery agent of the present invention enhances cellular uptake. According to certain embodiments of the invention, a composition comprising purified polypeptide expressed from a mammalian cell line and a delivery agent is formulated as large porous particles for aerosol administration.

  Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, for example, transmucosal administration includes detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the purified polypeptide or protein and the delivery agent are formulated into ointments, salves, gels, or creams generally known in the art.

  The compositions can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

  In one embodiment, the composition is prepared with a carrier that protects the polypeptide or protein from rapid elimination from the body, such as a sustained release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are from Alza Corporation and Nova Pharmaceuticals, Inc. Can also be purchased from Liposomal suspensions (including liposomes targeted to infected cells having monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

  It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for the subject to be treated, each unit as desired in connection with the required pharmaceutical carrier. Contains a predetermined amount of active polypeptide or protein calculated to produce a therapeutic effect.

  Polypeptides or proteins expressed according to the present invention may be administered at various intervals and for various periods as needed, for example, about 1-10 weeks, 2-8 weeks, about 3-7 weeks, about once a week. It can be administered at 4, 5, or 6 weeks. Those skilled in the art will recognize that a particular factor, including but not limited to the severity of the disease or disorder, the previous treatment, the subject's overall health and / or age, and other diseases present may be effective for the subject. It should be understood that this may affect the dose and timing required to treat the patient. In general, treatment of a subject with a polypeptide or protein described herein may include a single treatment or, in many cases, a series of treatments. In addition, the appropriate dose may depend on the potency of the polypeptide or protein and may be tailored to a particular recipient, for example by administering increasing doses until a preselected desired response is achieved. It is understood that there is. The specific dose level for any particular animal subject is the activity of the specific polypeptide or protein used, the subject's age, weight, overall health, sex, and diet, time of administration, route of administration, excretion rate It will be understood that it can depend on a variety of factors, including any drug combination, as well as the degree of modulation or expression or activity.

  The present invention includes the use of the compositions of the present invention to treat non-human animals. Accordingly, the dosage and method of administration may be selected according to veterinary pharmacology and medical known principles. The guidelines are for example Adams, R .; (Eds.), Veterinary Pharmacology and Therapeutics, 8th edition, Iowa State University Press, ISBN: 0813817439, 2001.

  The pharmaceutical compositions of the invention can be included in a container, pack, or dispenser along with instructions for administration.

Kits The present invention also provides kits that contain components useful for the preparation of cell culture media that contain the iron-containing compositions described herein. Such kits can be particularly useful for increasing cell density, viability, and / or titer in cell culture.

In some embodiments, such kits of the invention include one or more reagents for making an initial cell culture medium. Such kits may include one or more reagents useful for supplementing known cell culture media (eg, hormones and / or other growth factors, inorganic ions sodium, chloride, calcium, magnesium, etc. And buffers such as phosphates, vitamins, nucleosides or nucleotides, trace elements, amino acids, lipids, or glucose or other energy sources). The kit of the present invention may include a separate composition comprising iron (eg, FeSO ₄ , Fe citrate, Fe-transferrin, Fe chloride, Fe nitrate, Fe-EDTA, Fe (NO ₃ ) ₃ , FeCl ₂ Or FeCl ₃ ). In some embodiments, a kit of the invention includes a separate composition comprising iron that provides an appropriate concentration (eg, in the range between 100 μM to 5 mM) in the cell culture media described herein. included. The kit of the present invention also includes instructions (eg, instructions) for adding a separate composition of iron to the initial cell culture medium at one or more points during the cell culture process described herein. May be contained.

  The components of the kit of the present invention may be provided in individual containers, and multiple such containers may be provided together in a common housing.

  The invention is illustrated in further detail by the following non-limiting examples. The examples are provided for illustration only and should not be construed to limit the scope of the invention.

Example 1
Addition of Fe to fed-batch cultures after day 3 This experiment was performed with significant addition of Fe after 3 days of fed-batch culture of cells producing therapeutic proteins, resulting in significant cell density, viability and titer. Demonstrate the increase.

  CHO cells, cell line 1, were cultured in a known composition enriched medium using a fed-batch culture process. High concentrations of Fe (eg, 100 μM-5 mM) were added to the cell culture after day 3, resulting in various Fe concentrations (eg, 0 mM, 0.1 mM, 1.0 mM, 2.0 mM, or 5.0 mM). . In this experiment, ferric citrate was used and the cells were cultured in shake flasks. Cells were subjected to cell density, viability, and titer analysis at various time points throughout the cell culture process.

  Viable cell density was determined by CEDEX using a digital image recognition method. The effect of various doses of Fe addition after day 3 on the cell growth of exemplary cell line 1 is shown in FIG. As can be seen from FIG. 1, the addition of Fe after the third day increased the viable cell density compared to cells grown in the absence of iron.

  Cell viability was determined by CEDEX with trypan blue exclusion method. The effect of various doses of Fe addition after day 3 on the cell viability of exemplary cell line 1 is shown in FIG. As shown in FIG. 2, addition of Fe after the third day increased cell viability compared to cells grown in the absence of iron.

  In addition, the effect of Fe addition on cell viability at later stages of cell culture was also determined. Specifically, cell viability was determined on day 16 for cells grown in the presence or absence of iron. As shown in FIG. 3, the addition of Fe increased cell viability on the 16th day.

  The effect of addition of various doses of Fe after day 3 on antibody titer was also determined. Antibody titers were determined by the Protein A affinity HPLC method on day 16 for cells grown in the presence or absence of various concentrations of iron. As shown in FIG. 4, the addition of Fe increased the antibody titer on the 16th day.

  It was also observed that the optimal concentration of Fe addition after the third day is likely to be 0.3 mM to 1 mM. The addition of Fe on any day after the third day is effective. There is a possibility that supplying Fe in a plurality of parts is better than a single addition.

  Similar experiments were performed with the second exemplary cell line, cell line 2, and exemplary results are shown in FIG. As shown in FIG. 5, the addition of Fe improved cell density, viability and titer, especially at the late stage.

In addition to Fe citrate, the effects of other Fe complexes such as FeSO ₄ or Fe-transferrin were also tested. FIG. 6 summarizes exemplary results showing the effect of FeSO ₄ and Fe citrate addition after day 3 on viable cell density and viability on day 14 of fed-batch cell line 1. .

  Similar experiments were performed in both shake flask and bioreactor experiments, and the effect was the same. This effect was also observed with various products (such as various monoclonal antibodies).

  Thus, the beneficial effects of Fe addition are independent of cell line, cell culture scale, Fe source and product. It was also determined that the product quality was not affected by the high Fe concentration.

(Example 2)
Addition of Fe on day 6 CHO cells expressing Nanobodies were grown by fed-batch culture in shake flasks. Cells were initially cultured for 0-4 days at 37 ° C. in a 7% CO ₂ incubator and shifted to 31 ° C. after 4 days. On day 6, FeSO ₄ was added so that the iron concentration was 1 mM in the cell culture medium. Cells were subjected to cell density, viability, titer, lactate level and Qp analysis at various time points throughout the cell culture process as described in Example 1. Exemplary results are shown in FIGS. As shown in FIGS. 7-10, the addition of Fe improves viability (eg, 96% vs. 70% on day 15 as shown in FIG. 7), improves titer (eg, as shown in FIG. 3.2 g / L vs 1.5 g / L on day, improved Qp (eg 20 μg / e6 / day vs 10 μg / e6 / day as shown in FIG. 9), and reduced lactate levels (eg FIG. 10) Lactic acid was kept close to zero in Fe-added cell cultures, but not in control cultures without Fe and increased to 2.24 g / L on day 15). It showed significant advantages during cell culture.

(Example 3)
Fe Addition on Day 0 This experiment is designed to test whether the high concentration of Fe added on day 0 has an advantageous effect. Cells from a cell line producing a monoclonal antibody (clone 2.8) were cultured in a pH-controlled shake flask using 7% CO ₂ at about 120 rpm using an orbital shaker. The seeding density was 1.5 × 10 ⁶ cells / mL. Cells were cultured for 14 days. The basal medium was a chemically defined basal medium supplemented with amino acids containing 4 mM glutamine. The fed-batch medium is a chemically defined confederated fed medium of known composition. On day 0 or 9, Fe citrate or FeSO ₄ was added so that the iron concentration was 600 μM in the cell culture medium. Cells were subjected to cell density, viability, titer and Qp analysis at various time points throughout the cell culture process as described in previous examples. Exemplary results are shown in FIGS.

  Addition of Fe citrate on day 0 resulted in increased cell growth early in the cell culture, while addition of Fe on day 9 showed improved cell density later in the cell culture (FIG. 11). ). However, Fe citrate addition at day 0 does not appear to help maintain higher viability at the end of cell culture. For example, as shown in FIG. 12, the viability on day 14 of the cell culture supplemented with 600 μM Fe citrate on day 0 is approximately 75%, which is 14% of the control culture without Fe addition. Comparable to 74-80% viability on day. In contrast, cell viability of cell cultures supplemented with Fe on day 9 is 85-90%.

  Furthermore, as shown in FIG. 13, the Qp of the cell culture supplemented with Fe citrate on day 0 was lower than all other conditions throughout the culture.

  In some cases, toxicity was observed at high Fe concentrations when Fe was added at the start of cell culture. For example, Fe is added to cell line 1 on day 0 and an exemplary effect on cell viability on day 3 in shake flasks is shown in FIG. As can be seen from FIG. 14, toxicity was observed at Fe concentrations higher than 100 μM. Compared to the results shown in Examples 1 and 2, high concentrations of Fe added after day 0 were not only toxic, but also demonstrated advantages for cell culture including cell viability and cell growth. That is amazing.

Equivalents Those skilled in the art will appreciate or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to limit the above description, but is as set forth in the appended claims.

  In the claims, articles such as “a”, “an”, and “the” are one unless stated otherwise or otherwise apparent from the content. Or more than one. A claim or explanation that includes “or” between one or more members of a group, unless indicated otherwise, or unless otherwise apparent from the context A member is considered to be satisfied if one, more than one, or all of its members are present, used or otherwise related in a given product or process. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise associated with a given product or process. The invention includes embodiments in which multiple or all members of the group are present in, employed in, or otherwise associated with a given product or process. Further, the invention includes all variations, combinations, and permutations in which one or more restrictions, elements, clauses, descriptive terms, etc. from one or more of the recited claims are introduced within another claim. Will be understood to be included. For example, any claim that is dependent on another claim can be modified to include one or more restrictions found in any other claim that is dependent on the same base claim.

  Where elements are shown as a list, for example in a Markush group format, it should be understood that each subgroup of elements is also disclosed and any element (or elements) can be removed from the group. In general, when it is referred to that the present invention or aspects of the invention include certain elements, features, etc., certain embodiments of the invention or aspects of the invention consist of such elements, features, etc. It should be understood that it consists of it. For purposes of simplicity, those embodiments are not specifically described herein in this language. Note that the term “comprising” is intended to be open and allows the inclusion of additional elements or steps.

  If a range is given, end points are included. Further, unless otherwise specified, or unless otherwise apparent from the content or understanding of one of ordinary skill in the art, values expressed as ranges are clearly indicated otherwise from the content in different embodiments of the invention. It should be understood that any specific value or sub-range within the stated range can take up to one-tenth of the lower limit unit unless otherwise stated.

  Furthermore, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered known to those skilled in the art, they can be excluded even if the exclusion is not expressly stated herein. Any particular embodiment of the composition of the present invention (eg, any targeting moiety, any disease, disorder, and / or condition, for any reason, whether related to the presence of the prior art) Any linking agent, any method of administration, any therapeutic application, etc.) can be excluded from any one or more claims.

  The publications described above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

Incorporation of References All publications and patent documents cited in this application are incorporated in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein. Incorporated as a reference.

Claims (15)

(A) providing a cell in a cell culture medium for initiating a cell culture process, wherein the cell culture medium contains iron as a trace element;
(B) adding a composition containing iron to the cell culture medium during the cell culture process so that the concentration of iron in the cell culture medium increases over the period of the cell culture process, the iron containing the iron The composition is added on or after the third day of the cell culture process , and the concentration of iron in the cell culture medium after the addition of the iron-containing composition ranges between 100 μM and 5 mM. , A method of increasing cell density, viability, and / or titer in a cell culture medium. Compositions containing iron is selected FeSO _4, citric acid Fe, Fe- transferrin, chloride Fe, nitrate _{Fe, Fe-EDTA, Fe (} NO 3) 3, FeCl 2, FeCl 3 and combinations thereof The method of claim 1. The method according to claim 1 or 2 , wherein the concentration of iron in the cell culture medium after addition of the composition comprising iron is in the range between 300 μM and 1 mM. 4. The method according to any one of claims 1 to 3 , wherein the cell is a mammalian cell. The method of claim 4 , wherein the mammalian cell is a CHO cell. The method according to any one of claims 1 to 5 , wherein the cell culture step is a large-scale production culture step. The method of claim 6 , wherein the volume of the cell culture medium is at least 500 L. The method according to any one of claims 1 to 7 , wherein the cell carries a gene encoding a recombinant protein. The method according to claim 8 , wherein the recombinant protein is an antibody or a fragment thereof. 10. The method according to any one of claims 1 to 9 , further comprising the step of purifying the recombinant protein. Following the start of the cell culture process, look including the step of adding a composition comprising iron after the third day or, iron is, an amount such that the concentration in the cell culture medium in the range of between 100μM~5mM A method of preventing or delaying cell death in a cell culture , which is added in 12. The method of claim 11 , wherein iron is added in an amount such that the concentration in the cell culture medium ranges between 300 [mu] M to 1 mM. 13. A method according to any one of claims 11 or 12 , wherein the cell culture is a large scale production culture. 14. The method of claim 13 , wherein the cell culture volume is at least 500L. Compositions containing iron is selected FeSO _4, citric acid Fe, Fe- transferrin, chloride Fe, nitrate _{Fe, Fe-EDTA, Fe (} NO 3) 3, FeCl 2, FeCl 3 and combinations thereof 15. A method according to any one of claims 11 to 14 .

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